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Ca2+ modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca2+ mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca2+/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca2+-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.

A systematic forward geneticDrosophilascreen for electroretinogram mutants lacking synaptic transients identified thefuseless(fusl) gene, which encodes a predicted eight-pass transmembrane protein in the presynaptic membrane. Nullfuslmutants display >75% reduction in evoked synaptic transmission but, conversely, an approximately threefold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wild-typefusltransgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca2+concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca2+channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca2+channel domains required for efficient coupling of the Ca2+influx and synaptic vesicle exocytosis during neurotransmission.

A large number of mutants in the norpA gene, which encodes the phospholipase C (PLC) involved in Drosophila phototransduction, is available for the investigation of the effects of specific amino acid substitutions in PLC on biochemical and electrophysiological properties of these mutants. Of the 47 norpA mutants screened for PLC protein content, all but one (H43) displayed drastically decreased amounts of the protein suggesting that almost any mutational alteration has a deleterious effect on the integrity of the protein. Three new amino acids were identified in the catalytic domains X and Y that are important for PLC catalytic activity and the generation of photoreceptor responses (ERG). One of them was found substituted in H43, which showed a low specific PLC activity, a pronounced decrease in ERG sensitivity, and a wild-type-like response termination time. The response termination times obtained from three mutants was found to be approximately inversely proportional to the amount of PLC. In addition, we show that (i) the specific PLC activity is a key factor determining the photoreceptor sensitivity; (ii) the catalytic activity and response termination are separable functions of PLC; and (iii) a mutation in the putative G alpha-interacting C2 domain causes a preferentially strong defect in latency.

The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel.

The trp and trpl genes are thought to encode two classes of light-activated ion channels in Drosophila . A previous report indicated that a null trpl mutant does not display any mutant phenotype. This lack of detectable mutant phenotypes made it difficult to suggest functions for the transient receptor potential-like (TRPL) channel in photoreceptor responses. Here, the properties of trpl photoreceptor responses were studied by using electroretinogram (ERG) and intracellular recording techniques in combination with light stimuli of relatively long durations. Distinct mutant phenotypes were detectable under these conditions. These consisted of a reduced sustained component, oscillations superimposed on the response, a poststimulus hyperpolarization, and altered adaptation properties to dim background light. Comparison of photoreceptor responses obtained from wild type, trp , and trpl showed that the responses obtained from the trp and trpl null mutants did not sum up to that of the wild-type response. To explain the nonlinear summation at the peak of the response, [Reuss et al. (1997)][1] proposed that Ca2+ions entering through the TRP channel modulate TRP and TRPL channel activities differentially. However, nonlinear summation was present not only at the peak but throughout the duration of response. Two lines of evidence are presented to suggest that, in addition to the interaction proposed by [Reuss et al. (1997)][1], there are other forms of interactions between TRP and TRPL channels, probably involving the channel proteins themselves. [1]: #ref-18

TheDrosophila trpgene encodes a light-activated Ca2+channel subunit, which is a prototypical member of a novel class of channel proteins. Previously identifiedtrpmutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. We report herein a new mutant,TrpP365, that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. We show that, in spite of its unusual phenotypes,TrpP365is atrpallele because aTrpP365transgene induces the mutant phenotype in a wild-type background, and a wild-typetrptransgene in aTrpP365background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause theTrpP365phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels ofTrpP365are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration inDrosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors inDrosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain.

We have identified and analyzed a Drosophila melanogaster gene that encodes a chloride channel subunit (DrosGluCl-α) previously shown to function as a glutamategated chloride channel in an in vitro expression system. Sequence analysis of several cDNAs corresponding to the gene revealed sequence diversity in their open reading frames at seven specific sites. Site-specific A-to-G variations between cDNA and genomic sequences, consistent with RNA editing, were detected at five nucleotide positions. In addition, sequence variations among cDNA clones consistent with alternative splicing of mRNA were found at two different sites. In the 5′ region, two small adjacent exons, containing similar but distinct modular sequences, are alternatively incorporated into the mature mRNA. In the 3′ region, alternative splicing generates a variant encoding a protein with four additional amino acids just upstream of the fourth transmembrane domain. Combinations of RNA editing and alternative splicing can lead to extensive diversification of transcripts. These results give the first example of RNA editing in neurotransmitter-gated chloride channel genes or of alternative splicing in a glutamate-gated chloride channel gene of Drosophila.

Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.

The Drosophila ninaA gene encodes photoreceptor-specific cyclophilin thought to play a critical role in rhodopsin folding or transport during its synthesis or maturation in the most abundant subclass of photoreceptors. Cyclophilins comprise a highly conserved family of proteins which are the primary targets of the potent immunosuppressive drug, cyclosporin A (CsA), and which display peptidyl prolyl cis-trans-isomerase (PPIase) activity. In an attempt to identify mammalian cyclophilins with properties similar to the NinaA protein, a probe derived from the ninaA cDNA was used to screen bovine retinal cDNA libraries. The screen identified two major alternatively spliced forms of cDNA that would encode proteins containing a region of high homology to other cyclophilins and that are expressed specifically in the retina. These proteins represent a new class of cyclophilins with novel structural features and greatly reduced PPIase and CsA binding activities in comparison to other known cyclophilins. Tissue in situ hybridization and immunolocalization of the proteins showed that the RNA and protein products are expressed in photoreceptors as well as other retinal neurons. However, among photoreceptors, the proteins are found predominantly in cones. Thus, mammalian retinas do contain cyclophilins that are retina- specifically and photoreceptor class-preferentially expressed. The results suggest that, in cones, the main function of these proteins is, like the NinaA protein, to facilitate proper folding or intracellular transport of opsins.

We have isolated, by interspecies hybridization, two classes of Drosophila cDNA each encoding a different guanylate cyclase (GC). One of them encodes an alpha subunit homolog of soluble GC, designated DGC alpha 1, and the other encodes a receptor-type GC, designated DrGC. The dgc alpha 1 cDNA encodes a protein of 676 amino acids and maps to 99B. In situ hybridization to adult tissue sections showed that dgc alpha 1 mRNA is found mainly in the cell bodies of the optic lobe, central brain, and thoracic ganglia. The DGC alpha 1 protein was also localized primarily to the nervous system by immunocytochemical staining, consistent with results of in situ hybridization. However, no detectable expression of this protein was found in the retina. The other class of cDNA, drgc, maps to 76C and encodes a 1525-amino acid protein displaying structural features similar to other known receptor-type guanylate cyclases. However, it has a C-terminal 430 amino acid region that has no homology to any known proteins. drgc RNA is expressed at low levels throughout development and in adult heads and bodies. In situ hybridizations to adult tissue sections showed that drgc mRNA is expressed in a wide range of tissues, including the optic lobe, central brain, thoracic ganglia, digestive tract, and the oocyte.

Our objective is to present a comprehensive view of the PDA (prolonged depolarizing afterpotential)-defective Drosophila mutants, nina's and ina's, from the discussion of the PDA and the PDA-based mutant screening strategy to summaries of the knowledge gained through the studies of mutants generated using the strategy. The PDA is a component of the light-evoked photoreceptor potential that is generated when a substantial fraction of rhodopsin is photoconverted to its active form, metarhodopsin. The PDA-based mutant screening strategy was adopted to enhance the efficiency and efficacy of ERG (electroretinogram)-based screening for identifying phototransduction-defective mutants. Using this strategy, two classes of PDA-defective mutants were identified and isolated, nina and ina, each comprising multiple complementation groups. The nina mutants are characterized by allele-dependent reduction in the major rhodopsin, Rh1, whereas the ina mutants display defects in some aspects of functions related to the transduction channel, TRP (transient receptor potential). The signaling proteins that have been identified and elucidated through the studies of nina mutants include the Drosophila opsin protein (NINAE), the chaperone protein for nascent opsin (NINAA), and the multifunctional protein, NINAC, required in multiple steps of the Drosophila phototransduction cascade. Also identified by the nina mutants are some of the key enzymes involved in the biogenesis of the rhodopsin chromophore. As for the ina mutants, they led to the discovery of the scaffold protein, INAD, responsible for the nucleation of the supramolecular signaling complex. Also identified by the ina mutants is one of the key members of the signaling complex, INAC (ePKC), and two other proteins that are likely to be important, though their roles in the signaling cascade have not yet been fully elucidated. In most of these cases, the protein identified is the first member of its class to be so recognized.

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol-3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant-negative or deleterious gain-of-function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain the efficacy of synaptic function.

This review recounts the early history of Drosophila phototransduction genetics, covering the period between approximately 1966 to 1979. Early in this period, the author felt that there was an urgent need for a new approach in phototransduction research. Through inputs from a number of colleagues, he was led to consider isolating Drosophila mutants that are defective in the electroretinogram. Thanks to the efforts of dedicated associates and technical staff, by the end of this period, he was able to accumulate a large number of such mutants. Particularly important in this effort was the use of the mutant assay protocol based on the "prolonged depolarizing afterpotential." This collection of mutants formed the basis of the subsequent intensive investigations of the Drosophila phototransduction cascade by many investigators.

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.

Drosophilavisual signaling, a G-protein-coupled phospholipase Cβ (PLCβ)-mediated mechanism, is regulated by eye-protein kinase C (PKC) that promotes light adaptation and fast deactivation, most likely via phosphorylation of inactivation no afterpotential D (INAD) and TRP (transient receptor potential). To reveal the critical phosphatases that dephosphorylate INAD, we used several biochemical analyses and identified protein phosphatase 2A (PP2A) as a candidate. Importantly, the catalytic subunit of PP2A, microtubule star (MTS), is copurified with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in threemtsheterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC [INAC (inactivation no afterpotential C]in vivo, we performed ERG recordings. We discovered thatinaCP209was semidominant, becauseinaCP209heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect ofinaCP209heterozygotes was rescued by themtsXE2258heterozygous background. In contrast,mtsXE2258failed to modify the severe deactivation ofnorpAP16, indicating that MTS does not modulate NORPA (no receptor potential A) (PLCβ). Together, our results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC, restoring the fast deactivation kineticsin vivo. We thus propose that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD.

SummaryIn Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.

SUMMARY Division of labor in honey bee colonies is influenced by the foraging gene (Amfor), which encodes a cGMP-dependent protein kinase (PKG). Amfor upregulation in the bee brain is associated with the age-related transition from working in the hive to foraging for food outside, and cGMP treatment (which increases PKG activity)causes precocious foraging. We present two lines of evidence in support of the hypothesis that Amfor affects division of labor by modulating phototaxis. We first show that a subset of worker bees involved in the removal of corpses from the hive had forager-like brain levels of Amfor brain expression despite being middle aged; age-matched food-handlers, who do not leave the hive to perform their job, had low levels of Amforexpression. This finding suggests that occupations that involve working outside the hive are associated with high levels of Amfor in brain. Secondly, foragers were much more positively phototactic than hive bees in a laboratory assay, and cGMP treatment caused a precocious onset of positive phototaxis. The cGMP effect was not due to a general increase in behavioral activity; cGMP treatment had no effect on locomotor activity under either constant darkness or a light:dark regime. The cGMP effect also was not due to changes in circadian rhythmicity; cGMP treatment had no effect on age at onset of locomotor circadian rhythmicity or the period of rhythmicity. The effects of Amfor on phototaxis are not related to peripheral processing;electroretinogram analysis revealed no effect of cGMP treatment on photoreceptor activity and no differences between untreated hive bees and foragers. The cAMP/PKA pathway does not appear to be playing a similar role to cGMP/PKG in the honey bee; cAMP treatment did not affect phototaxis and gene expression analysis revealed task-related differences only for the gene encoding the regulatory subunit, but not the catalytic subunit, of PKA. Our findings implicate one neural process associated with honey bee division of labor that can be affected by naturally occurring changes in the expression of Amfor.

The Drosophila norpA gene encodes a phosphatidylinositol-specific phospholipase C (PI-PLC) expressed predominantly in photoreceptors and involved in phototransduction. However, no direct role for a phospholipase C in vertebrate phototransduction has been identified to date. Recently, we reported the isolation and characterization of bovine cDNAs encoding PI-PLC isoforms expressed predominantly in the retina and with higher homology to the NorpA protein than to any other known PI-PLC. Here, we present evidence that the norpA-homologous bovine retinal PI-PLCs, although found in other retinal neurons as well, are found in cones but not in rods. The results suggest that the phototransduction cascade in cones may utilize phospholipase C in addition to phosphodiesterase.

The trp gene of Drosophila encodes a subunit of a class of Ca 2+ -selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate “store-operated Ca 2+ entry,” which is important in Ca 2+ homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Here, we report on the Drosophila inaF gene, which encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The inaF mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the trp gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that inaF mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function.

In insects, histamine is found both in the peripheral nervous system (PNS) and in the CNS and is known to function as a fast neurotransmitter in photoreceptors that have been shown to express selectively thehdcgene. This gene codes for histidine decarboxylase (HDC), the enzyme for histamine synthesis. Fast neurotransmission requires the efficient removal of the transmitter from the synaptic cleft. Here we identify inDrosophilaphoto- and mechanoreceptors a histamine uptake mechanism that can restore the function of these receptors in mutants unable to synthesize histamine. When apparent null mutants for thehdcgene imbibe aqueous histamine solution or are genetically “rescued” by a transgene ubiquitously expressing histidine decarboxylase under heat-shock control, sufficient amounts of histamine selectively accumulate in photo- and mechanoreceptors to generate near-normal electrical responses in second-order visual interneurons and qualitatively to restore wild-type visual and mechanosensory behavior. This strongly supports the proposal that histamine functions as a fast neurotransmitter also in a certain class of mechanoreceptors. A set of CNS-intrinsic neurons that in the wild type contain high concentrations of histamine apparently lacks this uptake mechanism. We therefore speculate that histamine of intrinsic neurons may function as a neuromodulator rather than as a fast transmitter.

Cyclophilins are ubiquitous and abundant proteins that exhibit peptidyl prolyl cis-trans isomerization (PPlase) activity in vitro. Their functions in vivo, however, are not well understood. Two new retinal cyclophilin isoforms, types I and II, are highly expressed in cone photoreceptors of the vertebrate retina. Type-II cyclophilin is identical to RanBP2, a large protein that binds the GTPase Ran. Here we report that two contiguous domains in RanBP2, Ran-binding domain 4 (RBD4) and cyclophilin, act in concert as a chaperone for the opsin molecule of the red/green-sensitive visual pigment of a dichromatic vertebrate. In Drosophila, the cyclophilin NinaA is expressed in all photoreceptors and is required for the expression of only a subset of opsins. The molecular basis of these photoreceptor class-specific effects and the functions of NinaA and other cyclophilins in vivo remain unclear. Unlike NinaA, which forms a stable complex with opsin from retinular cells R1-6, we find that the cyclophilin domain of RanBP2 does not bind opsin directly; rather, it augments and stabilizes the interaction between red/green (R/G) opsin and the RBD4 domain. This involves a cyclophilin-mediated modification of R/G opsin, possibly involving proline isomerization. The RBD4-cyclophilin supradomain of RanBP2, therefore, is a form of vertebrate chaperone of defined substrate specificity, which may be involved in the processing and/or transport of long-wavelength opsin in cone photoreceptor cells.

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

Drosophila mutants of a single complementation group with defective on-/off-transients of the electroretinogram (ERG) were found to be deficient in synthesis of the photoreceptor transmitter, histamine, in a gene-dosage dependent manner, suggesting that the gene identified by the mutants (hdc) might be the structural gene for Drosophila histidine decarboxylase (HDC). A rat HDC cDNA was used to isolate a Drosophila homolog which shows approximately 60% sequence identity with mammalian HDCs over a region of 476 amino acids. In RNA blots, the Drosophila homolog detects four transcripts that are expressed primarily in the eye and are severely reduced in hdc mutants. The cloned Drosophila cDNA hybridizes to the 46F region of the chromosome, to which hdc mutations have been mapped, and rescues the hdc mutant phenotype in transgenic flies generated by P element-mediated germline transformation. The results thus show that the Drosophila homolog corresponds to the histidine decarboxylase gene, identified by the hdc mutants, and that mutations in the gene disrupt photoreceptor synaptic transmission.

Mitochondrial porins, also know as VDACs (voltage-dependent anion channels), play an important role in regulating energy metabolism, apoptosis, and the transport of metabolites across the mitochondrial outer membrane. So far three distinct isoforms of VDAC (VDAC1-3) have been reported in vertebrates, but their functions remain unknown. The annotation database of the Drosophila melanogaster genome sequence has identified four genes (porin, CG17137, CG17139, and CG17140) encoding different isoforms of VDACs. We identified post-translational modifications of PORIN that are specific to D. melanogaster eyes. We also identified the P-element insertion in the porin gene, porinG2294, that is homozygous viable whereas all the porin mutants previously reported are homozygous lethal at the pupal stage. The mutant does not show any defects in fly morphology, survival, and photoreceptor structure. The mutant, however, produces

A rapid electrical potential, which we have named the M-potential, can be obtained from the Drosophila eye using a high energy flash stimulus. The potential can be elicited from the normal fly, but it is especially prominent in the mutant norp AP12 (a phototransduction mutant), particularly if the eye color pigments are genetically removed from the eye. Several lines of evidence suggest that the M-potential arises from photoexcitation of long-lived metarhodopsin. Photoexcitation of rhodopsin does not produce a comparable potential. The spectral sensitivity of the M-potential peaks at about 575 nm. The M-potential pigment (metarhodopsin) can be shown to photoconvert back and forth with a "silent pigment(s)" absorbing maximally at about 485 nm. The silent pigment presumably is rhodopsin. These results support the recent spectrophotometric findings that dipteran metarhodopsin absorbs at much longer wavelengths than rhodopsin. The M-potential probably is related to the photoproduct component of the early receptor potential (ERP). Two major differences between the M-potential and the classical ERP are: (a) Drosophila rhodopsin does not produce a rapid photoresponse, and (b) an anesthetized or freshly sacrificed animal does not yield the M-potential. As in the case of the ERP, the M-potential appears to be a response associated with a particular state of the fly visual pigment. Therefore, it should be useful in in vivo investigations of the fly visual pigment, about which little is known.

Small potential fluctuations ("bumps"), boyh spontaneous and light induced, can be recorded intracellularly from the photoreceptors of Drosophila melanogaster. Statistical analyses of these bumps in the spectral range, 400-600 nm, lead to the following interpretations; (a) For weak stimuli at least, these bumps are the quantal units of the receptor potential. (b) Quanta of various wavelengths, when effectively absorbed, will elicit bumps of the same average size. (c) The spectral sensitivity of the receptor potential appears to have its origin in the relative efficiency of quantum bump production at different wavelengths, and not in the intrinsic difference in the properties of bumps produced by quanta of differenct wavelengths.

1. Dark-adaptation of rod photoreceptors has been studied in the isolated axolotl (Ambystoma mexicanum) retina by intracellular recordings. Rod responsiveness was greatly reduced immediately after a 30 sec partial bleach, but partially recovered with time in the dark. 2. In parallel spectrophotometric measurements using isolated retinas, regeneration of the rod pigment could not be detected after a 30 sec bleach. 3. During rod dark-adaptation, the response of a rod to a given stimulus increased in amplitude, duration, and rate of rise but did not recover completely to the dark-adapted values. Response latency was lengthened immediately after a bleach but ultimately returned to the dark-adapted level. 4. The time courses of dark-adaptation determined on the basis of the intensity of a stimulus needed to evoke a response having a criterion amplitude, a criterion duration, or a criterion rate of rise were similar. On the other hand changes in latency of the response and magnitude of the saturated amplitude followed different time courses. Change in log threshold was found to be related to change in saturated amplitude by an exponential function during dark-adaptation. 5. After bleaching 10% or less of the rod pigment, the kinetics of both recovery of log threshold and decrease in absorbance at 400 nm (metarhodopsin II+free retinal) could be described by two concurrent first-order processes having similar time constants. However, after bleaching more than 10% of the rod pigment, changes in sensitivity and absorbance did not follow parallel time courses. 6. Metarhodopsin III cannot be solely responsible for setting the axolotl rod sensitivity since rod thresholds decrease monotonically during dark-adaptation whereas meta III concentration reaches a peak 3 min after the bleach and decreases thereafter.

A polypeptide present in Drosophila eye homogenates was identified as opsin. This polypeptide pI 7.8, with Mr 39,000 is a retina-specific protein. It has the spectral characteristics of rhodopsin contained in the R1-6 photoreceptors and decreases in amount with vitamin A deprivation. It contains a chromophore derived from vitamin A and linked to the protein moiety by a Schiff base. Moreover, the polypeptide identified corresponds to a retina-specific polypeptide that was shown previously to undergo light-dependent phosphorylation in living flies. These results indicate that many properties of Drosophila rhodopsin do not differ significantly from those reported for rhodopsins of other organisms. However, the isoelectric point of Drosophila opsin is considerably more basic than those reported for vertebrate rhodopsins.

The time-course of light-induced changes in membrane voltage and resistance were measured in single photoreceptors in eyecup preparations of Gekko gekko. A small circular stimulus directed toward the impaled receptor produced membrane hyperpolarization. Application of a steady annular light to the receptor periphery resulted in diminution of the receptor's response to the stimulus. The effects of illumination of the surrounding receptors were isolated by directing a small, steady desensitizing light to the impaled receptor and then applying a peripheral stimulus. Brief stimuli produced a transient decrease in resistance with rapid onset and offset, a time-course similar to that of the response diminution. For some cells a depolarization that coincided with the resistance decrease was seen. During illumination with prolonged stimuli the resistance decrease was followed by a slow increase. After offset resistance rose transiently above the original value and then returned slowly to its original value. The slow resistance changes were not accompanied by changes in membrane voltage. The response diminution, resistance decrease, and depolarization were not observed in retinas treated with aspartate or hypoxia. It is therefore concluded that these effects are mediated by horizontal cells. The diminution is achieved by shunting the receptor potential and may play a role in field adaptation.

A Drosophila melanogaster gene (dgo) encoding a G protein alpha subunit has been isolated by screening genomic and adult head cDNA libraries using bovine transducin alpha subunit cDNA as probe. The gene, which maps to 47A on the second chromosome, encodes two proteins which are both 354 amino acids long but differ in seven amino acids in the amino-terminal region. The deduced amino acid sequences of the two proteins are 81% identical to that of a rat Go alpha subunit. Analysis of genomic clones revealed that there are eight coding exons and that the putative transcripts for the two proteins differ in the 5'-noncoding regions and the first coding exons but share the remaining six coding exons. The arrangement of two different 5'-noncoding regions on the gene suggests that two different promoters regulate the expression of the transcripts encoding the two proteins. RNA blot analysis detected three transcripts: a 3.9-kilobase (kb) transcript found at all stages of development; a 5.4-kb transcript present predominantly in adult heads; and a 3.4-kb transcript present only in adult bodies. In situ hybridizations of a cDNA probe to adult tissue sections showed that the gene is expressed abundantly in neuronal cell bodies in the brain, optic lobe, and thoracic ganglia.

The electroretinogram of the dipteran compound eye in response to an intense flash contains an early, diphasic potential that has been termed the M potential. Both phases of the M potential arise from the photostimulation of metarhodopsin. The early, corneal-negative component, the M1, can be recorded intracellularly in the photoreceptors and has properties similar to the classical early receptor potential (ERP). The M1 is resistant to cold, anaesthesia, and anoxia and has no detectable latency. It depends on flash intensity and metarhodopsin fraction in the manner predicted for a closed, two-state pigment system, and its saturation is shown to correspond to the establishment of a photoequilibrium in the visual pigment. On the other hand, the dominant, corneal-positive component, the M2, does not behave like an ERP. It arises, not in the photoreceptors, but deeper in the retina at the level of the lamina, and resembles the on-transient of the electroretinogram in its reversal depth and sensitivity to cooling or CO2. The on-transient, which is present over a much wider range of stimulus intensity than the M potential, has been shown to arise from neurons in the lamina ganglionaris. Visual mutants in which the on-transient is absent or late are also defective in the M2. It is proposed that the M2 and the on-transient arise from the same or similar groups of second-order neurons, and that the M2 is a fast laminar response to the depolarizing M1 in the photoreceptors, just as the on-transient is a fast laminar response to the depolarizing late receptor potential. Unlike the M1, the M2 is not generally proportional to the amount of metarhodopsin photoconverted, and the M2 amplitude is influenced by factors, such as a steady depolarization of the photoreceptor, which do not affect the M1.

The dependence of pigment granule migration (PGM) upon the receptor potential was examined using several strains of electroretinogram (ERG)-defective mutants of Drosophila melanogaster. The mutants that have a defective lamina component but a normal receptor component of the ERG (no on-transient A [nonA] and tan) exhibited normal pigment granule migration. The mutants that have very small or no receptor potentials (certain no receptor potential A [norpA] alleles), on the other hand, exhibited no PGM. In the case of the temperature-sensitive norpA mutant, norpAH52, normal PGM was present at 17 degrees but not at 32 degrees C or above, corresponding to its electrophysiological phenotype. In the transient receptor potential (trp) mutant, whose receptor potential decays to the baseline within a few seconds during a sustained light stimulus, the pigment granules initially moved close to the rhabdomere when light was turned on but moved away after about 5 s during a sustained light stimulus. All these results lend strong support to the notion that PGM is initiated by a light-evoked depolarization of the receptor membrane, i.e., the receptor potential. However, under certain experimental conditions, the receptor potentials failed to induce PGM in the trp mutant. The depolarization of the receptor, thus, appears to be closely associated with PGM but is not a sufficient condition for PGM.

The second phase (negative peak) of the early receptor potential of cones has been studied in the all-cone eyes of the Mexican and antelope ground squirrels (Citellus mexicanus and Citellus leucurus) and compared with responses from the rod-dominant eyes of the rat and flying squirrel (Glaucomys volans). The responses obtained from the all-cone eyes tended to be smaller in amplitude, to have higher thresholds, and to be considerably more resistant to light adaptation than the responses from the rod-dominant eyes. The wave forms and time courses of the two types of responses were similar, although the cone potential tended to be less sensitive to temperature variations and its time constants tended to be shorter than those of the rod potential. The spectral sensitivity of the second phase of the early receptor potential of the Mexican ground squirrel closely follows the absorption spectrum of a Dartnall nomogram pigment having its absorption maximum at 540 mμ. Moreover, as in the case of the rat, the amplitude of the response appears to be linearly related to the amount of pigment bleached in a flash. Thus, in both all-rod and all-cone systems the early receptor potential appears to arise in the photoexcitation of the respective visual pigment and appears to be closely linked to the initial photochemical events. The similarity of the wave form, time course, and stimulus-response curves in the two systems suggests that the early receptor potential is produced by similar mechanisms in all-rod and all-cone systems.

Desensitisation of peripheral photoreceptors shown by blue-induced decrease in transmittance of Drosophila rhabdomeres

Fast electrical responses elicited from the transient intermediates in the bleaching of rhodopsin have been investigated in the retina of the albino rat. In the experiments we used two-flash stimuli triggered sequentially with a continuously variable time delay between them. At room temperature the potential is biphasic in wave-form. The dominant, corneal-positive component is preceded by a small, corneal-negative component. Cooling the retina to about 0 degrees C suppresses the positive component and isolates the faster, negative component. Experiments with color filters show that these two components display different spectral sensitivities and, hence, suggest that each of them is produced by a different photoproduct of bleaching.

A moderate light stimulus induced isoelectric point (pI) changes in three classes of retina-specific polypeptides (80, 49, and 39 kilodaltons) of Drosophila in vivo. When inorganic phosphate labeled with phosphorus-32 was fed to flies, the radioactive label was incorporated into these polypeptides during the pI changes, indicating light-induced phosphorylation of the polypeptides. A 1-millisecond flash induced a detectable amount of phosphorylation in the 80- and 49-kilodalton polypeptides within 3 seconds. These results, and our previous results with norpA mutants, suggest that phosphorylation of these two polypeptides may be involved in some early stages of photoreceptor excitation or its modulation.

The ninaC gene is one of eight nina (neither inactivation nor afterpotential) genes identified from mutations that drastically reduce the amount of rhodopsin in the compound eye of Drosophila melanogaster. The gene has been cytogenetically localized to the 27E-28B region of the second chromosome. NaDodSO4/PAGE analysis of eye proteins of flies carrying one, two, or three copies of the ninaC region shows that two eye-specific proteins of molecular weight 170,000 and 130,000 display a strong dependence on the dosage of the ninaC gene, although the dependence is evident only when the dosage is decreased and not when it is increased. All mutations in the ninaC gene studied to date have pronounced effects on these two polypeptides. These results suggest that the ninaC locus encodes these two polypeptides. Ultrastructural studies show that the polypeptides encoded by ninaC are very likely to be important components of the cytoskeletal structure of rhabdomeral microvilli.

The Drosophila photoreceptor potential is thought to be composed of discrete unit potentials called bumps. The steady-state receptor potential and the accompanying voltage fluctuations were recorded intracellularly under steady illumination. The occurrence rate, effective amplitude, and duration of the bumps were deduced by assuming a shot noise model. Over a wide range of light intensity, the duration of bumps remained essentially constant (25-30 ms). Below the saturation intensity for the receptor potential, the bump rate was roughly proportional to the intensity, and the adjustment of bumps to smaller size at higher intensity was mainly responsible for the nonlinear behavior of the receptor potential. The reduction in size of bumps at increasing light intensity was found to be due mainly to the diminishing magnitude of the bump current, and not to some other secondary effects. The bump rate saturated at about 3 x 105-106 events/s.

FOR a wide variety of photoreceptor cells, it has been shown that light alters the conductance of the cell membrane, giving rise to the voltage response of the cell1–4. It is generally accepted that the primary event in the photo-excitation process is a light-induced isomerisation of the photopigment chromophore. The mechanisms linking this isomerisation to the conductance change remain unknown. It has been suggested recently that the transduction process may depend on oxidative metabolism. Intracellular recordings from photoreceptors of the honeybee (A pis)5, horseshoe crab (Limulus)5 and barnacle (Balanus)—observed recently by one of the authors (A.M.) in collaboration with R. Lantz—have shown that the light-induced conductance increase during illumination can be reversibly blocked by anoxia. Neither the site nor the mechanism of the action of anoxia on the transduction process is known. The light-induced spectral transitions of the photopigments are known, however, to take place under a variety of conditions including anoxia6–10.

Thirty genetic alterations, which involve the 4BC region of the Drosophila X chromosome, have been induced by ionizing radiation or by an endogenous mutator element. These mutations were recovered by screening for reversion of the dominant mutants Oce and Qd or for induction of the recessive mutants bi and rb. Among the 23 mutants generated by ionizing radiation, 20 have proven to be cytologically detectable chromosomal aberrations. Seven additional unique aberrations were generated in the Uc mutator strain. In total, 22 cytologically detectable deficiencies, 3 translocations, 1 inversion, 1 transposition, and 3 cytologically normal mutants have been recovered. Complementation analysis has permitted the cytogenetic localization of eight genes in the 4BC region. The mei-9 locus has been assigned to region 4B4-6, because this function is carried by Df(1)rb41 but not by Df(1)biD1. The norpA locus has been placed in the 4B6-C1 region based on its location between the distal breakpoints of Df(1)biD2 and Df(1)rb41. The genes lac, Qd, bi, and omb are localized to bands 4C5,6, rb to 4C6 and amb to 4C7,8. With one exception the complementation analysis has also permitted a determination of the linear sequence of these genes. This cytogenetic localization of these loci will facilitate the cloning and molecular analysis of genes controlling a key function in DNA repair and recombination (mei-9), and two fundamental neural functions (norpA and omb).

Mutations in the ninaA gene of Drosophila severely reduce the amount of rhodopsin specifically in R1-6 photoreceptors. Isolation of the ninaA gene by chromosomal walking revealed that it is expressed only in the eye and encodes a 237-amino acid polypeptide that shows strong sequence similarity to cyclophilin, a putative molecular target for cyclosporine A, a potent immunosuppressant used in human organ transplantations. Unlike most cyclophilins characterized to date, the ninaA-encoded protein has a putative signal sequence and a transmembrane domain. Each of the three ehtyl methanesulfonate-induced ninaA mutant alleles analyzed shows a single nucleotide change in the mRNA coding region leading to either a nonsense or a missense mutation. We find no evidence that the ninaA-encoded protein is directly involved in phototransduction. The only detectable mutant phenotype that correlates with the severity of molecular defects in the three mutants is the amount of depletion of R1-6 rhodopsin. The above results and the recent findings that cyclophilin is a peptidylprolyl cis-trans-isomerase suggest that the ninaA-encoded protein may be required for proper folding and stability of R1-6 rhodopsin.

A Drosophila mutant (ninaAP228) that is low in rhodopsin concentration but identical to the wild-type fly in photoreceptor morphology has been isolated. R1-6 photoreceptors of the mutant differ from those of wild type in that (a) the prolonged depolarizing afterpotential (PDA) is absent, (b) concentrations of rhodopsin and opsin are substantially reduced, and (c) intramembrane particle density in the membranes of the rhabdomeres is low. Each of these traits is mimicked by depriving wild-type flies of vitamin A. The ninaAP228 mutation differs from vitamin A deprivation in that in the mutant (a) the rhabdomeric membrane particle density is reduced only in the R1-6 photoreceptors and not in R7 or R8, (b) the PDA can be elicited from the R7 photoreceptors, and (c) photoconversion of R1-6 rhodopsin to metarhodopsin by ultraviolet (UV) light is considerably more efficient than in vitamin A-deprived flies. The absorption properties of the mutant rhodopsin in the R1-6 photoreceptors appear to be identical to those of wild type as judged from rhodopsin difference spectra. The results suggest that the mutation affects the opsin, rather than the chromophore, component of rhodopsin molecules in the R1-6 photoreceptors. The interaction between the chromophore and R1-6 opsin, however, appears to be normal.

RANDOMLY occurring, discrete voltage fluctuations (quantum bumps) have been recorded in photoreceptors of several arthropods1–4. It is thought that these bumps sum to produce the receptor potential5,6. We have investigated the relationship between photoexcitation of the visual pigment and the production of quantum bumps using a mutant of Drosophila melanogaster—a third chromosome recessive mutant described by Cosens and Manning7, in which the electroretinogram decays to baseline during a strong, prolonged stimulus. We will call this mutant the transient receptor potential mutant (trp).

The ionic mechanism of horizontal cell potentials was investigated in the isolated retina of the axolotl Ambystoma mexicanum. The membrane potentials of both receptors and horizontal cells were recorded intracellularly while the ionic composition of the medium flowing over the receptor side of the retina was changed. The membrane potential of the horizontal cell is highly depender side of the retina was changed. The membrane potential of the horizontal cell is highly dependent on the extracellular concentration of sodium. When the external ion concentration of either chloride or potassium was changed independently of the other, there were shifts in the membrane potential of the horizontal cell which could not be explained by changes in the equilibrium potential of these ions. If the external concentrations of both potassium and chloride ions were varied so that the product of their external concentrations did not change, the shift in the membrane potential of the horizontal cell was in the direction predicted by the Nernst equation. The results are consistent with the suggestion that in the dark the receptors release a synaptic transmitter which increases primarily the sodium conductance of the horizontal cell postsynaptic membrane.